Combination therapies using anti-metabolites and agents that target tumor-associated stroma or tumor cells

ABSTRACT

The present invention provides, inter alia, methods for treating or ameliorating the effects of a disease, such as cancer, in a subject. The methods include: administering to a subject in need thereof (a) a therapeutically effective amount of an agent selected from an anti-metabolite agent or analog thereof and combinations thereof; and (b) a therapeutically effective amount of a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody contains: (i) a heavy chain variable region (VH), which includes an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and (ii) a light chain variable region (VL), which includes an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. Compositions, including pharmaceutical compositions, and kits for treating diseases, such as cancer, are also provided herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Patent Application Ser. No.62/009,380, filed on Jun. 9, 2014 which application is incorporated byreference herein in its entirety.

FIELD OF INVENTION

The present invention provides, inter alia, methods for treating orameliorating the effects of a disease, such as cancer, in a subject. Themethods include administering to a subject in need thereof (i)anti-metabolite agents or analogs thereof, and (ii) TEM8 antibodies orantigen binding fragments thereof. Compositions, includingpharmaceutical compositions, and kits for treating diseases, such ascancer, are also provided herein.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acidsequences that have been filed concurrently herewith as sequence listingtext file “0385323.txt,” file size of 37 KB, created on Jun. 5, 2015.The aforementioned sequence listing is hereby incorporated by referencein its entirety pursuant to 37 C.F.R. §1.52(e)(5).

BACKGROUND OF THE INVENTION

Targeting tumor-associated stroma, especially tumor associatedvasculature, is considered a promising approach to cancer therapy.Various classes of chemotherapeutics directed toward tumor vasculaturehave been developed, including anti-angiogenic agents and vasculardisrupting agents, the former affecting neovascularization and thelatter targeting existing blood vessels that supply tumors withnutrients and oxygen. Though these therapies are widely used,particularly in cases of metastatic cancer, they are hampered by theirtoxicity and off-target effects against healthy vasculature. Thus, thereexists, inter alia, a need for additional therapeutics that are lesstoxic and work in combination with current agents to suppress tumorgrowth by targeting the tumor-associated stroma or the tumor cellsthemselves. The present invention is directed to meeting these and otherneeds.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for treating orameliorating the effects of a disease in a subject. The methodcomprises:

administering to a subject in need thereof

(a) a therapeutically effective amount of an anti-metabolite agent oranalog thereof; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region (VH), which comprises an amino        acid sequence selected from the group consisting of SEQ ID NO:1,        SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and    -   (ii) a light chain variable region (VL), which comprises an        amino acid sequence selected from the group consisting of SEQ ID        NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

Another embodiment of the present invention is a method for treating orameliorating the effects of a disease in a subject. The methodcomprises:

administering to a subject in need thereof

(a) a therapeutically effective amount of an anti-metabolite agent oranalog thereof; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region, which comprises an amino acid        sequence selected from SEQ ID NO:5; and    -   (ii) a light chain variable region, which comprises an amino        acid sequence selected from SEQ ID NO:6.

A further embodiment of the present invention is a method for treatingor ameliorating the effects of a disease in a subject. The methodcomprises:

administering to a subject in need thereof

(a) a therapeutically effective amount of 5-fluorouracil; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region, which comprises an amino acid        sequence selected from SEQ ID NO:5; and    -   (ii) a light chain variable region, which comprises an amino        acid sequence selected from SEQ ID NO:6.

An additional embodiment of the present invention is a composition fortreating or ameliorating the effects of a disease in a subject. Thecomposition comprises:

(a) a therapeutically effective amount of an anti-metabolite agent oranalog thereof; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region (VH), which comprises an amino        acid sequence selected from the group consisting of SEQ ID NO:1,        SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and    -   (ii) a light chain variable region (VL), which comprises an        amino acid sequence selected from the group consisting of SEQ ID        NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

Another embodiment of the present invention is a composition fortreating or ameliorating the effects of a disease in a subject. Thecomposition comprises:

(a) a therapeutically effective amount of an anti-metabolite agent oranalog thereof; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region, which comprises an amino acid        sequence selected from SEQ ID NO:5; and    -   (ii) a light chain variable region, which comprises an amino        acid sequence selected from SEQ ID NO:6.

A further embodiment of the present invention is a composition fortreating or ameliorating the effects of a disease in a subject. Thecomposition comprises:

(a) a therapeutically effective amount of 5-fluorouracil; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region, which comprises an amino acid        sequence selected from SEQ ID NO:5; and    -   (ii) a light chain variable region, which comprises an amino        acid sequence selected from SEQ ID NO:6.

An additional embodiment of the present invention is a pharmaceuticalcomposition. The pharmaceutical composition comprises any of thecompositions disclosed herein and a pharmaceutically acceptable diluentor carrier.

Another embodiment of the present invention is a kit. The kit comprisesany of the compositions or pharmaceutical compositions disclosed hereinpackaged together with instructions for its use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D show results from an efficacy study of TEM8 antibodies in aHCT116 colorectal carcinoma xenograft model. Treatment was initiatedwhen tumor volumes were approximately 60-80 mm³. TEM 8 antibody (hIgG1)was dosed at 5 and 15 mg/kg, three times per week for three weeks.Bevacizumab was dosed at 5 mg/kg, two times per week for three weeks.FIG. 1A shows a box plot of group median tumor volume distribution onday 21. FIG. 1B shows mean tumor growth over the time course of thestudy. FIG. 1C shows individual times to the endpoint for each mouse inthe study. FIG. 1D shows percent body weight change for each group overthe course of the study.

FIG. 2 shows a line graph of tumor volume in the UACC melanoma model.Treatments were initiated when tumor volumes were approximately 50 mm³.TEM 8 antibodies (hIgG1) were dosed at 15 mg/kg, three times per week,for 3-4 weeks.

FIG. 3 shows a line graph of tumor volume over time in the HCT116 coloncancer model. The M825 antibody and drug-conjugated M825-MMAE were given2 times per week for 3 weeks.

FIG. 4 shows a line graph of tumor volume over time in an OVCAR3 ovariancancer model. TEM8 antibody M825 and antibody-drug conjugate (ADC)M825-MMAE were given 2 times per week for 3.5 weeks. The 10 mg/kg ADCgroup was redosed on Day 56, 2 times per week for the remainder of thestudy.

FIG. 5 shows a line graph of tumor volume over time in the MDA-MB231breast cancer model. M825 and M825-MMAE were given 2 times per week for3 weeks.

FIGS. 6A-D show results from a study of treatment with TEM8 M830antibody, bevacizumab, paclitaxel, and combinations thereof in the H460non-small cell lung carcinoma xenograft model. FIG. 6A shows a timecourse of mean tumor growth throughout the study. FIG. 6B shows aKaplan-Meier survival plot. FIG. 6C shows individual times to endpointfor mice in each group. FIG. 6D shows percent body weight change foreach group throughout the study.

FIGS. 7A-E show results from a study of treatment with M830,gemcitabine, cisplatin, radiation, and combinations thereof in a H460non-small cell lung carcinoma xenograft model. FIG. 7A shows a timecourse of mean tumor growth throughout the study. FIG. 7B shows the timecourse with median tumor growth of each group. FIG. 7C shows aKaplan-Meier survival plot. FIG. 7D shows individual times to endpointfor mice in each group. FIG. 7E shows percent body weight change foreach group throughout the study.

FIG. 8 shows a line graph of tumor volume over time from a study ofM830, bevacizumab, irinotecan, and combinations thereof in a DLD-1colorectal adenocarcinoma xenograft model.

FIG. 9 shows a line graph of tumor volume over time from a study oftreatment with combinations of M830, bevacizumab, irinotecan, and thecombination of all three treatments in a DLD-1 colorectal adenocarcinomaxenograft model.

FIG. 10 shows a Kaplan-Meier survival plot demonstrating the effect ofM830 antibody in mice in a MC38 colon carcinoma liver metastasis model.

FIGS. 11A-B show the effect of M825 on 3T3 fibroblasts in the presenceof TGFb-1. FIG. 11A shows pictures of the cultured cells over 48 hoursand FIG. 11B is a bar graph quantifying the lattice area of the cells.

FIGS. 12A-B show the effect of M830 Fc mutation on antibody-dependentcell-mediated cytotoxicity (ADCC). FIG. 12A is a schematic showing theantibody binding in the ADCC assay and FIG. 12B is a line graphdemonstrating that the Fc mutant form of M830 has significantly enhancedbinding to FcγRIIIa (CD16a).

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is a method for treating orameliorating the effects of a disease in a subject. The methodcomprises:

administering to a subject in need thereof

(a) a therapeutically effective amount of an anti-metabolite agent oranalog thereof; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region (VH), which comprises an amino        acid sequence selected from the group consisting of SEQ ID NO:1,        SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and    -   (ii) a light chain variable region (VL), which comprises an        amino acid sequence selected from the group consisting of SEQ ID        NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

As used herein, the terms “treat,” “treating,” “treatment” andgrammatical variations thereof mean subjecting an individual subject toa protocol, regimen, process or remedy, in which it is desired to obtaina physiologic response or outcome in that subject, e.g., a patient. Inparticular, the methods and compositions of the present invention may beused to slow the development of disease symptoms or delay the onset ofthe disease or condition, or halt the progression of diseasedevelopment. However, because every treated subject may not respond to aparticular treatment protocol, regimen, process or remedy, treating doesnot require that the desired physiologic response or outcome be achievedin each and every subject or subject population, e.g., patientpopulation. Accordingly, a given subject or subject population, e.g.,patient population may fail to respond or respond inadequately totreatment.

As used herein, the terms “ameliorate”, “ameliorating” and grammaticalvariations thereof mean to decrease the severity of the symptoms of adisease in a subject.

As used herein, a “subject” is a mammal, preferably, a human. Inaddition to humans, categories of mammals within the scope of thepresent invention include, for example, farm animals, domestic animals,laboratory animals, etc. Some examples of farm animals include cows,pigs, horses, goats, etc. Some examples of domestic animals includedogs, cats, etc. Some examples of laboratory animals include primates,rats, mice, rabbits, guinea pigs, etc.

In the present invention, cyclooxygenase-2 (COX-2) inhibitors (COXIBs),non-steroidal anti-inflammatory drugs (NSAIDs), and/or prostaglandin E2(PGE2) synthase inhibitors may be administered to the subject inaddition to or as a replacement for the anti-metabolite agent. COXIBsaccording to the present invention include, but are not limited to,AAT-076 (AskAt), celecoxib (Pfizer), valdecoxib (Pfizer), rofecoxib(Merck), nimesulide, 4-hydroxynimesulide, flosulide, L475L337, bromfenac(InSite Vision), KIT-302 (Kitov), polmacoxib (Pacificpharma), etodolac(Pfizer), parecoxib sodium (Pfizer), etoricoxib (Merck), lumiracoxib(Novartis), VA-964 (Rottapharm Madaus), iguratimod (Toyama), andmeloxicam (Boehringer Ingelheim). NSAIDs according to the presentinvention include, but are not limited to, diclofenac (Novartis),diflunisal (Merck), etodolac (Almirall Limited), fenoprofen (RanbaxyPharmaceuticals), flurbiprofen (Pfizer), ibuprofen (Pfizer),indomethacin (Lundbeck, Inc.), ketoprofen (Wyeth), ketorolac (Hospira),mefenamic acid (Shionogi, Inc.), meloxicam (Abbott Laboratories),nabumetone (GlaxoSmithKline), naproxen (Bayer), oxaprozin (Pfizer),piroxicam (Pfizer), sulindac (Mutual Pharmaceutical Company, Inc.),AAT-076 (AskAt), carbasalate calcium (Bristol-Myers Squibb), rimazoliummetilsulfate (Chinoin), isonixin (Hermes), lysine clonixinate (Laplex),alminoprofen (Recordati), lornoxicam (Takeda), fosfosal (Uriach),nepafenac (Novartis), diclofenac (Novartis), and tolmetin(Ortho-McNeil-Janssen Pharmaceuticals, Inc.). PGE2 synthase inhibitorsaccording to the present invention include, but are not limited to,NS-398, sulindac sulphide, leukotriene C4, GRC-27864 (Glenmark), OX-MPI(Orexo), PF-04693627 (Pfizer), and those described in, for example, He,et al, 2013.

As used herein, an “anti-metabolite agent” is a substance that reducesor inhibits a cell's use of a chemical that is part of normalmetabolism. As used herein, an “analog” is a compound that isstructurally similar to, e.g., the anti-metabolite agent, and that alsoreduces or inhibits the growth of cancer cells. Non-limiting examples ofanti-metabolite agents or analogs thereof according to the presentinvention include antifolates, purine inhibitors, pyrimidine inhibitors,and combinations thereof.

As used herein, an “antifolate” is a substance that alters, reduces, orinhibits the use of folic acid (vitamin B₉) by cells. Non-limitingexamples of antifolates include methotrexate (DuraMed Pharmaceuticals,Inc.), pemetrexed (Eli Lilly), pralatrexate (Spectrum Pharmaceuticals),aminopterin (Sigma Aldrich), pharmaceutically acceptable salts thereof,and combinations thereof.

As used herein, a “purine” is a compound that contains a fusedsix-membered and a five-membered nitrogen-containing ring. Non-limitingexamples of purines that are important for cellular metabolism includeadenine, guanine, hypoxanthine, and xanthine. A “purine inhibitor” is asubstance that alters, reduces or suppresses the production of a purineor the use of a purine by a cell. Non-limiting examples of purineinhibitors include methotrexate (DuraMed Pharmaceuticals, Inc.),pemetrexed (Eli Lilly), hydroxyurea (Bristol-Myers Squibb),2-mercaptopurine (Sigma-Aldrich), 6-mercaptopurine (Sigma-Aldrich),fludarabine (Ben Venue Laboratories), clofarabine (Genzyme Corp.),nelarabine (GlaxoSmithKline), pralatrexate (Spectrum Pharmaceuticals),6-thioguanine (Gate Pharmaceuticals), forodesine (BioCrystPharmaceuticals), pentostatin (Bedford Laboratories), sapacitabine(Cyclacel Pharmaceuticals, Inc.), aminopterin (Sigma Aldrich),azathioprine (GlaxoSmithKline), pharmaceutically acceptable saltsthereof, and combinations thereof.

As used herein, a “pyrimidine” is a compound that contains asix-membered nitrogen-containing ring. Non-limiting examples ofpyrimidines that are important for cellular metabolism include uracil,thymine, cytosine, and orotic acid. A “pyrimidine inhibitor” is asubstance that alters, reduces, or suppresses the production of apyrimidine or the use of a pyrimidine by the a cell. Non-limitingexamples of pyrimidine inhibitors include 5-fluorouracil (TocrisBioscience), tegafur (LGM Pharma), capecitabine (Xeloda) (Roche),cladribine (LGM Pharma), gemcitabine (Eli Lilly), cytarabine (BedfordLaboratories), decitabine (Eisai Inc.), floxuridine (BedfordLaboratories), 5-azacytidine (Pharmion Pharmaceuticals), doxifluridine(Cayman Chemicals), thiarabine (Access Pharmaceuticals), troxacitabine(SGX Pharmaceuticals), raltitrexed (AstraZeneca), carmofur (Santa CruzBiotechnology, Inc.), 6-azauracil (MP Biomedicals, LLC),pharmaceutically acceptable salts thereof, and combinations thereof.

In a preferred aspect of the present invention, the anti-metaboliteagent or an analog thereof is selected from the group consisting of5-fluorouracil (Tocris Bioscience), tegafur (LGM Pharma), capecitabine(Xeloda) (Roche), cladribine (LGM Pharma), methotrexate (DuraMedPharmaceuticals, Inc.), pemetrexed (Eli Lilly), hydroxyurea(Bristol-Myers Squibb), 2-mercaptopurine (Sigma-Aldrich),6-mercaptopurine (Sigma-Aldrich), fludarabine (Ben Venue Laboratories),gemcitabine (Eli Lilly), clofarabine (Genzyme Corp.), cytarabine(Bedford Laboratories), decitabine (Eisai Inc.), floxuridine (BedfordLaboratories), nelarabine (GlaxoSmithKline), pralatrexate (SpectrumPharmaceuticals), 6-thioguanine (Gate Pharmaceuticals), 5-azacytidine(Pharmion Pharmaceuticals), doxifluridine (Cayman Chemicals), forodesine(BioCryst Pharmaceuticals), pentostatin (Bedford Laboratories),sapacitabine (Cyclacel Pharmaceuticals, Inc.), thiarabine (AccessPharmaceuticals), troxacitabine (SGX Pharmaceuticals), raltitrexed(AstraZeneca), aminopterin (Sigma Aldrich), carmofur (Santa CruzBiotechnology, Inc.), azathioprine (GlaxoSmithKline), 6-azauracil (MPBiomedicals, LLC), pharmaceutically acceptable salts thereof, andcombinations thereof.

As used herein, an “antibody” and “antigen-binding fragments thereof”encompass naturally occurring immunoglobulins (e.g., IgM, IgG, IgD, IgA,IgE, etc.) as well as non-naturally occurring immunoglobulins,including, for example, single chain antibodies, chimeric antibodies(e.g., humanized murine antibodies), heteroconjugate antibodies (e.g.,bispecific antibodies), Fab′, F(ab′)₂, Fab, Fv, and rlgG. See, e.g.,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby et al., 1998. As used herein, an “antigen-binding fragment”is a portion of the full length antibody that retains the ability tospecifically recognize the antigen, as well as various combinations ofsuch portions.

Non-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly, or can be obtained,for example, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein by reference.These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies, are well knownto those skilled in the art (Winter and Harris, Immunol. Today14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow andLane, supra, 1988; Hilyard et al., Protein Engineering: A practicalapproach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed.(Oxford University Press 1995); each of which is incorporated herein byreference).

Full length antibodies can be proteolytically digested down to severaldiscrete, functional antibody fragments, which retain the ability torecognize the antigen. For example, the enzyme papain can be used tocleave a full length immunoglobulin into two Fab fragments and an Fcfragment. Thus, the Fab fragment is typically composed of two variabledomains and two constant domains from the heavy and light chains. The Fvregion is usually recognized as a component of the Fab region andtypically comprises two variable domains, one from each of the heavy(V_(H), “heavy chain variable region”, as used herein) and light (V_(L)“light chain variable region”, as used herein) chains. The enzyme pepsincleaves below the hinge region, so a F(ab′)₂ fragment and a pFc′fragment is formed. F(ab′)₂ fragments are intact antibodies that havebeen digested, removing the constant (Fc) region. Two Fab′ fragments canthen result from further digestion of F(ab′)₂ fragments. Examples ofantigen-binding fragments include, but are not limited to, Fv, Fab,Fab′, Fab′-SH, F(ab′)₂, diabodies, tribodies, scFvs, and single-domainantibodies (dAbs).

Typically, a full length antibody has at least one heavy and at leastone light chain. Each heavy chain contains a variable domain (V_(H)) andtypically three or more constant domains (C_(H)1, C_(H)2, C_(H)3, etc.),while each light chain contains a variable domain (V_(L)) and a constantdomain C_(L). Light and heavy chain variable regions contain four“framework” regions interrupted by three hypervariable regions, alsocalled “complementarity-determining regions” or “CDRs”. The extent ofthe framework regions and CDRs have been defined. See, e.g., Kabat etal., U.S. Dept. of Health and Human Services, Sequences of Proteins ofImmunological Interest (1983) and Chothia et al., J. Mol. Biol.196:901-917 (1987). The sequences of the framework regions of differentlight or heavy chains are relatively conserved within a species. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody, whereas a V_(L) CDR1 is the CDR1 from thevariable domain of the light chain of the antibody.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. The modifier “monoclonal” indicatesthe character of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made, e.g., by the hybridoma method firstdescribed by Kohler et al., Nature 256: 495 (1975), and as modified bythe somatic hybridization method as set forth above; or may be made byother recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

Additional types of antibodies that may be part of the monoclonalantibodies of the present invention include, but are not limited to,chimeric, humanized, and human antibodies. For application in man, it isoften desirable to reduce immunogenicity of antibodies originallyderived from other species, like mouse. This can be done by constructionof chimeric antibodies, or by a process called “humanization”. In thiscontext, a “chimeric antibody” is understood to be an antibodycomprising a domain (e.g. a variable domain) derived from one species(e.g. mouse) fused to a domain (e.g. the constant domains) derived froma different species (e.g. human).

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions are those of a human immunoglobulin sequence.The humanized antibody optionally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol2:593-596 (1992)). Humanization can be essentially performed, e.g.,following the method of Winter and co-workers (Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-3′27 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody.

Furthermore, technologies have been developed for creating antibodiesbased on sequences derived from the human genome, for example by phagedisplay or using transgenic animals (see, e.g., WO 90/05144; D. Marks,H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D. Griffiths and G.Winter (1991) “By-passing immunisation. Human antibodies from V-genelibraries displayed on phage.” J. Mol. Biol., 222, 581-597; Knappik etal., J. Mol. Biol. 296: 57-86, 2000; S. Carmen and L. Jermutus,“Concepts in antibody phage display”. Briefings in Functional Genomicsand Proteomics 2002 1(2):189-203; Lonberg N, Huszar D. “Human antibodiesfrom transgenic mice”. Int Rev Immunol. 1995; 13(1):65-93; Bruggemann M,Taussig M J. “Production of human antibody repertoires in transgenicmice”. Curr Opin Biotechnol. 1997 August; 8(4):455-8.). Such antibodiesare “human antibodies” in the context of the present invention.

As used herein, a “recombinant” antibody is any antibody whoseproduction involves expression of a non-native DNA sequence encoding thedesired antibody structure in an organism. In the present invention,recombinant antibodies include tandem scFv (taFv or scFv₂), diabody,dAb₂/VHH₂, knob-into-holes derivatives, SEED-IgG, heteroFc-scFv,Fab-scFv, scFv-Jun/Fos, Fab′-Jun/Fos, tribody, DNL-F(ab)₃, scFv₃-CH1/CL,Fab-scFv₂, IgG-scFab, IgG-scFv, scFv-IgG, scFv₂-Fc, F(ab′)₂-scFv₂,scDB-Fc, scDb-CH3, Db-Fc, scFv₂-H/L, DVD-Ig, tandAb, scFv-dhlx-scFv,dAb₂-IgG, dAb-IgG, dAb-Fc-dAb, and combinations thereof.

Variable regions of antibodies are typically isolated as single-chain Fv(scFv) or Fab fragments. ScFv fragments are composed of V_(H) and V_(L)domains linked by a short 10-25 amino acid linker. Once isolated, scFvfragments can be genetically linked with a flexible peptide linker suchas, for example, one or more repeats of Ala-Ala-Ala,Gly-Gly-Gly-Gly-Ser, etc. The resultant peptide, a tandem scFv (taFv orscFv₂) can be arranged in various ways, with V_(H)-V_(L) or V_(L)-V_(H)ordering for each scFv of the taFv. (Kontermann, R. E. In: BispecificAntibodies. Kontermann R E (ed.), Springer Heidelberg Dordrecht LondonNew York, pp. 1-28 (2011)).

As used herein, the term “epitope” refers to the portion of the antigenwhich is recognized by the antibody or antigen binding fragment. Asingle antigen (such as an antigenic polypeptide) may have more than oneepitope. Epitopes may be defined as structural or functional. Functionalepitopes are generally a subset of the structural epitopes and havethose residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents, whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents.

Portions of the preferred antibodies and nucleic acids encoding saidantibody portions of the present invention are listed below in Table 1.

TABLE 1 Antibody Sequences SEQ Nucleic Acid/ ID NO. Sequence NamePolypeptide Organism 1 M825 mAb VH Polypeptide Artificial sequence 2M825 mAb VL Polypeptide Artificial sequence 3 M822 mAb VH PolypeptideArtificial sequence 4 M822 mAb VL Polypeptide Artificial sequence 5 M830mAb VH Polypeptide Artificial sequence 6 M830 mAb VL PolypeptideArtificial sequence 7 M863 mAb VH Polypeptide Artificial sequence 8 M863mAb VL Polypeptide Artificial sequence 11 M825 mAb VH cDNA Nucleic acidArtificial sequence 12 M825 mAb VL cDNA Nucleic acid Artificial sequence13 M822 mAb VH cDNA Nucleic acid Artificial sequence 14 M822 mAb VL cDNANucleic acid Artificial sequence 15 M830 mAb VH cDNA Nucleic acidArtificial sequence 16 M830 mAb VL cDNA Nucleic acid Artificial sequence17 M863 mAb VH cDNA Nucleic acid Artificial sequence 18 M863 mAb VL cDNANucleic acid Artificial sequence

In one aspect of this embodiment, the disease is characterized bydifferential expression of a tumor endothelial marker 8 (TEM8) membraneantigen. As used herein, the terms “differential expression”,“differentially expressed”, and grammatical variations thereof meanchanges in the production levels of certain mRNA(s) and/or protein(s) intumor cells relative to normal cells. Differential expression includesupregulation and downregulation of gene(s). Preferably, the differentialexpression of TEM8 on, e.g. tumor endothelial cells, is sufficient forthe antibodies and/or antigen binding fragments thereof of the presentinvention to specifically target such cells, leaving normal,non-cancerous tissue substantially untouched.

Representative human TEM8 nucleic acid and polypeptide sequences areshown in Table 2 below.

TABLE 2 TEM8 Sequences SEQ ID Sequence Nucleic Other NO. NameAcid/Polypeptide Organism Information 9 TEM8 mRNA Nucleic acid HomoTranscript sapiens variant 1 10 TEM8 protein Polypeptide Homo Isoform 1sapiens precursor 19 TEM8 mRNA Nucleic acid Homo Transcript sapiensvariant 2 20 TEM8 protein Polypeptide Homo Isoform 2 sapiens precursor21 TEM8 mRNA Nucleic acid Homo Transcript sapiens variant 3 22 TEM8protein Polypeptide Homo Isoform 3 sapiens precursor

In another aspect of the invention, the disease is characterized bydifferential expression of tumor endothelial marker 8 (TEM8) membraneantigen on a tumor cell and/or a tumor stromal cell. As used herein,“tumor cells” comprise cells that have abnormal growth or division. Asused herein, “tumors” and “cancers” are used interchangeably. Tumors maybe benign or malignant. “Tumor stromal cells” include those cells thatare in a tumor cell's microenvironment and support the growth of tumorcells. Tumor vasculature is distinct from normal vasculature in thatseveral genes are differentially expressed in tumor-associated bloodvessels (St. Croix et al., 2000). One of these genes, tumor endothelialmarker 8 (TEM8) membrane antigen, is upregulated in the vasculature ofmalignant solid tumors, with limited expression in healthy tissues.

In another preferred embodiment, the disease is a cancer thatdifferentially expresses TEM8. More preferably, the cancer is selectedfrom the group consisting of kidney cancer, colon cancer, lung cancer,liposarcomas, brain cancer, breast cancer, melanoma, liver cancer, headand neck cancer, and prostate cancer.

In another aspect of this embodiment, the monoclonal antibody or antigenbinding fragment thereof comprises:

(1) a VH polypeptide encoded by:

(SEQ ID NO: 11) caggtccagctggtgcagtctggggctgaggtgaagaagcctgggacctcagtgaaggtctcctgcaaggttcctggatacaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacaacaaactacgcacagaagttccagggcagagtcacgattaccggggaggaatccacgagcacagtctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagagatacggactacatgtttgactactggggccagggaaccctggtcaccgtgagctc aand

(2) a VL polypeptide encoded by:

(SEQ ID NO: 12) tcttctgagctgactcaggaccctgttgtgtctgtggccttgggagagacagtcagtatcacatgccaaggagacaacctcagagacttttatgcaagctggtaccaacagaagccaggacaggcccctctactagtcatgtatggtaaaaacaggcggccctcagggatcccagaccgattctctggctccacctcaggaaacacactttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtagctcccgggacaacagtaagcatgtggtgttcggcggggggaccaaggtcaccgtccta.

In a further aspect of this embodiment, the monoclonal antibody orantigen binding fragment thereof comprises:

(1) a VH polypeptide encoded by:

(SEQ ID NO: 13) caggtccagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttctggatacaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagagatacggactacatgtttgactactggggccaggg aaccctggtcaccgtgagctcaand

(2) a VL polypeptide encoded by:

(SEQ ID NO: 14) tcttctgagctgactcaggaccctgttgtgtctgtggccttgggagagacagtcagtatcacatgccaaggagacaacctcagagacttttatgcaagctggtaccaacagaagccaggacaggcccctctactagtcatgtatggtaaaaacaggcggccctcagggatcccagaccgattctctggctccacctcaggaaacacactttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtagctcccgggacaacagtaagcatgtggtgttcggcggggggaccaaggtcaccgtccta.

In an additional aspect of this embodiment, the monoclonal antibody orantigen binding fragment thereof comprises:

(1) a VH polypeptide encoded by:

(SEQ ID NO: 15) gaggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccgtgagactctcctgtgcagcctctggattcaccttcagtacctatactatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcaattatctcaaatgatggaagcaataagtactacgcagaccccgtgaggggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctgtgtattactgtgtacgtggcagcagctggtatcgcggaaattggttcgacccctggggccagggaaccctggtcaccgtgagctcaand

(2) a VL polypeptide encoded by:

(SEQ ID NO: 16) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcgcttgccgggcaagtcagaccattagtaggtatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtctcatcaaggttcagtggcagtggatctgggacagagttcactctcaccatcagcagtctgcagcctgaagattttgcaacttatttctgtcaacagacttacagtcccccgatcaccttcggccaagggacacgactggagattaaacga.

In another aspect of this embodiment, the monoclonal antibody or antigenbinding fragment thereof comprises:

(1) a VH polypeptide encoded by:

(SEQ ID NO: 17) gaggtgcagctggtggagaccggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaccagtggtagcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcgggctgagatctgacgacactgccgtgtattactgtgtgagagatccgggttctcctaagtggctggccttcgacccctggggccagggcaccctggtcaccgtgagctca

(2) a VL polypeptide encoded by:

(SEQ ID NO: 18) gacatccagttgacccagtctccatcctccttgtctgcttctgtaggagacagagtcaccatcacttgccgggcaagtcgggccattagtaggtatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtctcatcaaggttcagtggcagtggatctgggacagagttcactctcaccatcagcagtctgcagcctgaagattttgcaacttatttctgtcaacagacttacagtcccccgatcaccttcggccaagggacacgactggagattaaacgt.

Another embodiment of the present invention is a method for treating orameliorating the effects of a disease in a subject. The methodcomprises:

administering to a subject in need thereof

(a) a therapeutically effective amount of an anti-metabolite agent oranalog thereof; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region, which comprises an amino acid        sequence selected from SEQ ID NO:5; and    -   (ii) a light chain variable region, which comprises an amino        acid sequence selected from SEQ ID NO:6.

Suitable and preferred anti-metabolite agents or analogs thereof,subjects, and diseases (including the characteristics of the diseasesand various types of cancers) are as disclosed herein.

A further embodiment of the present invention is a method for treatingor ameliorating the effects of a disease in a subject. The methodcomprises:

administering to a subject in need thereof

(a) a therapeutically effective amount of 5-fluorouracil; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region, which comprises an amino acid        sequence selected from SEQ ID NO:5; and    -   (ii) a light chain variable region, which comprises an amino        acid sequence selected from SEQ ID NO:6.

Suitable and preferred subjects, and diseases (including thecharacteristics of the diseases and various types of cancers) are asdisclosed herein.

An additional embodiment of the present invention is a composition fortreating or ameliorating the effects of a disease in a subject. Thecomposition comprises:

(a) a therapeutically effective amount of an anti-metabolite agent oranalog thereof; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region (VH), which comprises an amino        acid sequence selected from the group consisting of SEQ ID NO:1,        SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and    -   (ii) a light chain variable region (VL), which comprises an        amino acid sequence selected from the group consisting of SEQ ID        NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

Suitable and preferred anti-metabolite agents or analogs thereof,subjects, and diseases (including the characteristics of the diseasesand various types of cancers) are as disclosed herein.

In another aspect of this embodiment, the monoclonal antibody or antigenbinding fragment thereof comprises:

(1) a VH polypeptide encoded by:

(SEQ ID NO: 11) caggtccagctggtgcagtctggggctgaggtgaagaagcctgggacctcagtgaaggtctcctgcaaggttcctggatacaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacaacaaactacgcacagaagttccagggcagagtcacgattaccggggaggaatccacgagcacagtctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagagatacggactacatgtttgactactggggccaggg aaccctggtcaccgtgagctcaand

(2) a VL polypeptide encoded by:

(SEQ ID NO: 12) tcttctgagctgactcaggaccctgttgtgtctgtggccttgggagagacagtcagtatcacatgccaaggagacaacctcagagacttttatgcaagctggtaccaacagaagccaggacaggcccctctactagtcatgtatggtaaaaacaggcggccctcagggatcccagaccgattctctggctccacctcaggaaacacactttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtagctcccgggacaacagtaagcatgtggtgttcggcggggggaccaaggtcaccgtccta.

In a further aspect of this embodiment, the monoclonal antibody orantigen binding fragment thereof comprises:

(1) a VH polypeptide encoded by:

(SEQ ID NO: 13) caggtccagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttctggatacaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagagatacggactacatgtttgactactggggccaggg aaccctggtcaccgtgagctcaand

(2) a VL polypeptide encoded by:

(SEQ ID NO: 14) tcttctgagctgactcaggaccctgttgtgtctgtggccttgggagagacagtcagtatcacatgccaaggagacaacctcagagacttttatgcaagctggtaccaacagaagccaggacaggcccctctactagtcatgtatggtaaaaacaggcggccctcagggatcccagaccgattctctggctccacctcaggaaacacactttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtagctcccgggacaacagtaagcatgtggtgttcggcggggggaccaaggtcaccgtccta.

In an additional aspect of this embodiment, the monoclonal antibody orantigen binding fragment thereof comprises:

(1) a VH polypeptide encoded by:

(SEQ ID NO: 15) gaggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccgtgagactctcctgtgcagcctctggattcaccttcagtacctatactatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcaattatctcaaatgatggaagcaataagtactacgcagaccccgtgaggggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctgtgtattactgtgtacgtggcagcagctggtatcgcggaaattggttcgacccctggggccagggaaccctggtcaccgtgagctcaand

(2) a VL polypeptide encoded by:

(SEQ ID NO: 16) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcgcttgccgggcaagtcagaccattagtaggtatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtctcatcaaggttcagtggcagtggatctgggacagagttcactctcaccatcagcagtctgcagcctgaagattttgcaacttatttctgtcaacagacttacagtcccccgatcaccttcggccaagggacacgactggagattaaacga.

In another aspect of this embodiment, the monoclonal antibody or antigenbinding fragment thereof comprises:

(1) a VH polypeptide encoded by:

(SEQ ID NO: 17) gaggtgcagctggtggagaccggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaccagtggtagcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcgggctgagatctgacgacactgccgtgtattactgtgtgagagatccgggttctcctaagtggctggccttcgacccctggggccagggcaccctggtcaccgtgagctca

(2) a VL polypeptide encoded by:

(SEQ ID NO: 18) gacatccagttgacccagtctccatcctccttgtctgcttctgtaggagacagagtcaccatcacttgccgggcaagtcgggccattagtaggtatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtctcatcaaggttcagtggcagtggatctgggacagagttcactctcaccatcagcagtctgcagcctgaagattttgcaacttatttctgtcaacagacttacagtcccccgatcaccttcggccaagggacacgactggagattaaacgt.

Another embodiment of the present invention is a composition fortreating or ameliorating the effects of a disease in a subject. Thecomposition comprises:

(a) a therapeutically effective amount of an anti-metabolite agent oranalog thereof; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region, which comprises an amino acid        sequence selected from SEQ ID NO:5; and    -   (ii) a light chain variable region, which comprises an amino        acid sequence selected from SEQ ID NO:6.

Suitable and preferred anti-metabolite agents or analogs thereof,subjects, and diseases (including the characteristics of the diseasesand various types of cancers) are as disclosed herein.

A further embodiment of the present invention is a composition fortreating or ameliorating the effects of a disease in a subject. Thecomposition comprises:

(a) a therapeutically effective amount of 5-fluorouracil; and

(b) a therapeutically effective amount of a monoclonal antibody orantigen binding fragment thereof, wherein the monoclonal antibodycomprises:

-   -   (i) a heavy chain variable region, which comprises an amino acid        sequence selected from SEQ ID NO:5; and    -   (ii) a light chain variable region, which comprises an amino        acid sequence selected from SEQ ID NO:6.

Suitable and preferred subjects, and diseases (including thecharacteristics of the diseases and various types of cancers) are asdisclosed herein.

An additional embodiment of the present invention is a pharmaceuticalcomposition. The pharmaceutical composition comprises any of thecompositions disclosed herein and a pharmaceutically acceptable diluentor carrier. For the pharmaceutical compositions of the presentinvention, suitable and preferred anti-metabolite agents or analogsthereof, subjects, monoclonal antibodies or antigen binding fragmentsthereof (including those comprising specific SEQ ID NOs), and diseases(including the characteristics of the diseases and various types ofcancers) are as disclosed herein.

The pharmaceutical compositions according to the present invention maybe in a unit dosage form comprising both agents, namely (a), theanti-metabolite agent or an analog thereof, preferably 5-fluorouracil,(“(a) agent(s)”), and (b) a monoclonal antibody or antigen bindingfragment thereof according to the present invention (“(b) agent”). Inanother aspect of this embodiment, the agent(s) identified in (a) aboveis in a first unit dosage form and the agent identified in (b) above isin a second unit dosage form, separate from the first.

The agents identified in (a) and (b) above may be co-administered to thesubject, either simultaneously or at different times and in any order,as deemed most appropriate by a physician. If the (a) and (b) agents areadministered at different times, for example, by serial administration,the (a) agent(s) may be administered to the subject before the (b)agent. Alternatively, the (b) agent may be administered to the subjectbefore the (a) agent(s).

Another embodiment of the present invention is a kit. The kit comprisesany of the compositions or pharmaceutical compositions of the presentinvention packaged together with instructions for its use.

For use in the kits of the invention, compositions and pharmaceuticalcompositions comprising suitable and preferred anti-metabolite agents oranalogs thereof, subjects, monoclonal antibodies or antigen bindingfragments thereof (including those comprising specific SEQ ID NOs), anddiseases (including the characteristics of the diseases and varioustypes of cancers) are as set forth above. The kits may also includesuitable storage containers, e.g., ampules, vials, tubes, etc., for eachcomposition or pharmaceutical composition and other reagents, e.g.,buffers, balanced salt solutions, etc., for use in administering thecompositions or the pharmaceutical compositions to subjects. Thecompositions or the pharmaceutical compositions and other reagents maybe present in the kits in any convenient form, such as, e.g., in asolution or in a powder form. The kits may further include a packagingcontainer, optionally having one or more partitions for housing thecomposition or pharmaceutical composition and other optional reagents.

In the present invention, an “effective amount” or a “therapeuticallyeffective amount” of an agent, monoclonal antibody, or fragment thereofor a compound or composition disclosed herein is an amount of suchmaterial that is sufficient to effect beneficial or desired results asdescribed herein when administered to a subject. Effective dosage forms,modes of administration, and dosage amounts may be determinedempirically, and making such determinations is within the skill of theart. It is understood by those skilled in the art that the dosage amountwill vary with the route of administration, the rate of excretion, theduration of the treatment, the identity of any other drugs beingadministered, the age, size, and species of mammal, e.g., human patient,and like factors well known in the arts of medicine and veterinarymedicine. In general, a suitable dose of any active agent disclosedherein or a composition containing the same will be that amount of theactive agent or composition, which is the lowest dose effective toproduce the desired effect. The effective dose of an agent, monoclonalantibody, or fragment thereof or a compound or composition of thepresent invention may be administered as two, three, four, five, six ormore sub-doses, administered separately at appropriate intervalsthroughout the day.

A suitable, non-limiting example of a dosage of an anti-metabolite oranalog thereof, and a monoclonal antibody, or an antigen bindingfragment disclosed herein is from about 1 mg/kg to about 2400 mg/kg perday, such as from about 1 mg/kg to about 1200 mg/kg per day, includingfrom about 50 mg/kg to about 1200 mg/kg per day. Other representativedosages of such agents include about 5 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kgper day. The effective dose of anti-metabolite agents or analogsthereof, antibodies, and antibody fragments disclosed herein may beadministered as two, three, four, five, six or more sub-doses,administered separately at appropriate intervals throughout the day.

The compositions and pharmaceutical compositions of the presentinvention may be administered in any desired and effective manner: fororal ingestion, or as an ointment or drop for local administration tothe eyes, or for parenteral or other administration in any appropriatemanner such as intraperitoneal, subcutaneous, topical, intradermal,inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular,intravenous, intraarterial, intrathecal, or intralymphatic. Further, thecompositions and pharmaceutical compositions of the present inventionmay be administered in conjunction with other treatments. Eachcomposition and pharmaceutical composition of the present invention maybe encapsulated or otherwise protected against gastric or othersecretions, if desired.

The compositions and pharmaceutical compositions of the inventioncomprise one or more active ingredients in admixture with one or morepharmaceutically-acceptable diluents or carriers and, optionally, one ormore other compounds, drugs, ingredients and/or materials. Regardless ofthe route of administration selected, the agents/compounds of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington, The Science and Practice of Pharmacy (21st Edition,Lippincott Williams and Wilkins, Philadelphia, Pa.).

Pharmaceutically acceptable diluents or carriers are well known in theart (see, e.g., Remington, The Science and Practice of Pharmacy (21stEdition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and TheNational Formulary (American Pharmaceutical Association, Washington,D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, andsorbitol), starches, cellulose preparations, calcium phosphates (e.g.,dicalcium phosphate, tricalcium phosphate and calcium hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g., saline,sodium chloride injection, Ringer's injection, dextrose injection,dextrose and sodium chloride injection, lactated Ringer's injection),alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol),polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),organic esters (e.g., ethyl oleate and tryglycerides), biodegradablepolymers (e.g., polylactide-polyglycolide, poly(orthoesters), andpoly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils(e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut),cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones,talc, silicylate, etc. Each pharmaceutically acceptable diluent orcarrier used in a pharmaceutical composition of the invention must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the subject. Diluents orcarriers suitable for a selected dosage form and intended route ofadministration are well known in the art, and acceptable diluents orcarriers for a chosen dosage form and method of administration can bedetermined using ordinary skill in the art.

The compositions and pharmaceutical compositions of the invention may,optionally, contain additional ingredients and/or materials commonlyused in pharmaceutical compositions. These ingredients and materials arewell known in the art and include (1) fillers or extenders, such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid; (2)binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, sodium starch glycolate, cross-linked sodiumcarboxymethyl cellulose and sodium carbonate; (5) solution retardingagents, such as paraffin; (6) absorption accelerators, such asquaternary ammonium compounds; (7) wetting agents, such as cetyl alcoholand glycerol monostearate; (8) absorbents, such as kaolin and bentoniteclay; (9) lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, and sodium lauryl sulfate; (10)suspending agents, such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth;(11) buffering agents; (12) excipients, such as lactose, milk sugars,polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins,cocoa butter, starches, tragacanth, cellulose derivatives, polyethyleneglycol, silicones, bentonites, silicic acid, talc, salicylate, zincoxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13)inert diluents, such as water or other solvents; (14) preservatives;(15) surface-active agents; (16) dispersing agents; (17) control-releaseor absorption-delaying agents, such as hydroxypropylmethyl cellulose,other polymer matrices, biodegradable polymers, liposomes, microspheres,aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19)adjuvants; (20) wetting agents; (21) emulsifying and suspending agents;(22), solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan; (23) propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane; (24) antioxidants; (25) agents which render theformulation isotonic with the blood of the intended recipient, such assugars and sodium chloride; (26) thickening agents; (27) coatingmaterials, such as lecithin; and (28) sweetening, flavoring, coloring,perfuming and preservative agents. Each such ingredient or material mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the subject.Ingredients and materials suitable for a selected dosage form andintended route of administration are well known in the art, andacceptable ingredients and materials for a chosen dosage form and methodof administration may be determined using ordinary skill in the art.

Compositions and pharmaceutical compositions of the present inventionsuitable for oral administration may be in the form of capsules,cachets, pills, tablets, powders, granules, a solution or a suspensionin an aqueous or non-aqueous liquid, an oil-in-water or water-in-oilliquid emulsion, an elixir or syrup, a pastille, a bolus, an electuaryor a paste. These formulations may be prepared by methods known in theart, e.g., by means of conventional pan-coating, mixing, granulation orlyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared, e.g., bymixing the active ingredient(s) with one or morepharmaceutically-acceptable diluents or carriers and, optionally, one ormore fillers, extenders, binders, humectants, disintegrating agents,solution retarding agents, absorption accelerators, wetting agents,absorbents, lubricants, and/or coloring agents. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using a suitable excipient. A tablet may be made by compressionor molding, optionally with one or more accessory ingredients.Compressed tablets may be prepared using a suitable binder, lubricant,inert diluent, preservative, disintegrant, surface-active or dispersingagent. Molded tablets may be made by molding in a suitable machine. Thetablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient therein.They may be sterilized by, for example, filtration through abacteria-retaining filter. These compositions may also optionallycontain opacifying agents and may be of a composition such that theyrelease the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Any active ingredient of the invention can also be in microencapsulatedform.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Compositions and pharmaceutical compositions of the present inventionfor rectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more active ingredient(s) withone or more suitable nonirritating diluents or carriers which are solidat room temperature, but liquid at body temperature and, therefore, willmelt in the rectum or vaginal cavity and release the active compound.Compositions and pharmaceutical compositions of the present inventionwhich are suitable for vaginal administration also include pessaries,tampons, creams, gels, pastes, foams or spray formulations containingsuch pharmaceutically-acceptable carriers as are known in the art to beappropriate.

Dosage forms for topical or transdermal administration include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches,drops and inhalants. The active agent(s)/compound(s) may be mixed understerile conditions with a suitable pharmaceutically-acceptable diluentor carrier. The ointments, pastes, creams and gels may containexcipients. Powders and sprays may contain excipients and propellants.

Compositions and pharmaceutical compositions of the present inventionsuitable for parenteral administrations comprise one or moreagent(s)/compound(s) in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain suitable antioxidants,buffers, solutes which render the formulation isotonic with the blood ofthe intended recipient, or suspending or thickening agents. Properfluidity can be maintained, for example, by the use of coatingmaterials, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain suitable adjuvants, such as wetting agents, emulsifyingagents and dispersing agents. It may also be desirable to includeisotonic agents. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption.

In some cases, in order to prolong the effect of a drug (e.g., acomposition or a pharmaceutical composition of the present invention),it is desirable to slow its absorption from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility.

The rate of absorption of the active any active agent/composition of theinvention then depends upon its rate of dissolution which, in turn, maydepend upon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally-administered any active agent/compositionof the invention may be accomplished by dissolving or suspending theactive agent/composition in an oil vehicle. Injectable depot forms maybe made by forming microencapsule matrices of the active ingredient inbiodegradable polymers. Depending on the ratio of the active ingredientto polymer, and the nature of the particular polymer employed, the rateof active ingredient release can be controlled. Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue. The injectablematerials can be sterilized for example, by filtration through abacterial-retaining filter.

Any formulation of the invention may be presented in unit-dose ormulti-dose sealed containers, for example, ampules and vials, and may bestored in a lyophilized condition requiring only the addition of thesterile liquid diluent or carrier, for example water for injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the type described above.

ADDITIONAL DEFINITIONS

As used herein, terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymers.

The term “amino acid” means naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. An “amino acid analog”means compounds that have the same basic chemical structure as anaturally occurring amino acid, e.g., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. An “amino acid mimetic” means achemical compound that has a structure that is different from thegeneral chemical structure of an amino acid, but that functionssimilarly to a naturally occurring amino acid.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” used herein meanat least two nucleotides covalently linked together. Many variants of anucleic acid may be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequences. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be synthesized as a single strandedmolecule or expressed in a cell (in vitro or in vivo) using a syntheticgene. Nucleic acids may be obtained by chemical synthesis methods or byrecombinant methods.

The nucleic acid may also be a RNA such as a mRNA, tRNA, short hairpinRNA (shRNA), short interfering RNA (sRNA), double-stranded RNA (dsRNA),transcriptional gene silencing RNA (ptgsRNA), Piwi-interacting RNA,pri-miRNA, pre-miRNA, micro-RNA (miRNA), or anti-miRNA, as described,e.g., in U.S. patent application Ser. Nos. 11/429,720, 11/384,049,11/418,870, and 11/429,720 and Published International Application Nos.WO 2005/116250 and WO 2006/126040.

The nucleic acid may also be an aptamer, an intramer, or a spiegelmer.The term “aptamer” refers to a nucleic acid or oligonucleotide moleculethat binds to a specific molecular target. Aptamers are derived from anin vitro evolutionary process (e.g., SELEX (Systematic Evolution ofLigands by EXponential Enrichment), disclosed in U.S. Pat. No.5,270,163), which selects for target-specific aptamer sequences fromlarge combinatorial libraries. Aptamer compositions may bedouble-stranded or single-stranded, and may includedeoxyribonucleotides, ribonucleotides, nucleotide derivatives, or othernucleotide-like molecules. The nucleotide components of an aptamer mayhave modified sugar groups (e.g., the 2′—OH group of a ribonucleotidemay be replaced by 2′-F or 2′-NH₂), which may improve a desiredproperty, e.g., resistance to nucleases or longer lifetime in blood.Aptamers may be conjugated to other molecules, e.g., a high molecularweight carrier to slow clearance of the aptamer from the circulatorysystem. Aptamers may be specifically cross-linked to their cognateligands, e.g., by photo-activation of a cross-linker (Brody, E. N. andL. Gold (2000) J. Biotechnol. 74:5-13).

The term “intramer” refers to an aptamer which is expressed in vivo. Forexample, a vaccinia virus-based RNA expression system has been used toexpress specific RNA aptamers at high levels in the cytoplasm ofleukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA96:3606-3610).

The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA,or other left-handed nucleotide derivatives or nucleotide-likemolecules. Aptamers containing left-handed nucleotides are resistant todegradation by naturally occurring enzymes, which normally act onsubstrates containing right-handed nucleotides.

A nucleic acid will generally contain phosphodiester bonds, althoughnucleic acid analogs may be included that may have at least onedifferent linkage, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphosphoroamidite linkages and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those disclosed in U.S. Pat. Nos. 5,235,033 and5,034,506. Nucleic acids containing one or more non-naturally occurringor modified nucleotides are also included within the definition ofnucleic acid. The modified nucleotide analog may be located for exampleat the 5′-end and/or the 3′-end of the nucleic acid molecule.Representative examples of nucleotide analogs may be selected fromsugar- or backbone-modified ribonucleotides. It should be noted,however, that also nucleobase-modified ribonucleotides, i.e.ribonucleotides, containing a non-naturally occurring nucleobase insteadof a naturally occurring nucleobase such as uridines or cytidinesmodified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromouridine; adenosines and guanosines modified at the 8-position, e.g.8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; 0- andN-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The2′-OH-group may be replaced by a group selected from H, OR, R, halo, SH,SR, NH₂, NHR, NR₂ or CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyland halo is F, Cl, Br or I. Modified nucleotides also includenucleotides conjugated with cholesterol through, e.g., a hydroxyprolinollinkage as disclosed in Krutzfeldt et al., Nature (Oct. 30, 2005),Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent ApplicationPublication No. 20050107325. Modified nucleotides and nucleic acids mayalso include locked nucleic acids (LNA), as disclosed in U.S. PatentApplication Publication No. 20020115080. Additional modified nucleotidesand nucleic acids are disclosed in U.S. Patent Application PublicationNo. 20050182005. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments, to enhancediffusion across cell membranes, or as probes on a biochip. Mixtures ofnaturally occurring nucleic acids and analogs may be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Materials and Methods Mice

Unless otherwise noted, female immunodeficient athymic nude mice(Crl:NU(Ncr)-Foxn/nu, Charles River) were used at nine weeks old with abody weight (BW) range of about 15 to about 30 grams on Day 1 of thestudy.

In Vivo Implantation and Tumor Growth

Tumor cells were cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin G sodium,100 μg/mL streptomycin sulfate, and 25 μg/mL gentamicin. The tumor cellswere grown in tissue culture flasks in a humidified incubator at 37° C.,in an atmosphere of 5% CO₂ and 95% air.

On the day of tumor implant, each test mouse was injected with culturedtumor cells, and tumor growth was monitored as the average sizeapproached the target range. Tumors were measured in two dimensionsusing calipers, and volume was calculated using the formula:

Tumor Volume (mm³)=(w²×l)/2

where w=width and l=length, in mm, of the tumor. Tumor weight wasestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume. For all therapeutic studies, mice were sorted into groupscontaining the same average size tumors prior to initiation of therapy.

Controls

One group in each cohort received 1% vehicle IP three times per week toend, and served as the control group for calculation of % tumor growthdelay (TGD).

Endpoint and TGD Analysis

Tumors were measured using calipers twice per week, and each animal waseuthanized when its tumor reached the pre-determined tumor volumeendpoint described for each experiment (usually 2000 mm³) or on thefinal day, whichever came first. Animals that exited the study for tumorvolume endpoint were documented as euthanized for tumor progression(TP), with the date of euthanasia. The time to endpoint (TTE) foranalysis was calculated for each mouse by the following equation:

TTE=[log₁₀(endpoint volume)−b]/m

where TTE is expressed in days, endpoint volume is expressed in mm³, bis the intercept, and m is the slope of the line obtained by linearregression of a log-transformed tumor growth data set. The data setconsisted of the first observation that exceeded the endpoint volumeused in analysis and the three consecutive observations that immediatelypreceded the attainment of this endpoint volume. The calculated TTE wasusually less than the TP date, the day on which the animal waseuthanized for determination of tumor size. Animals with tumors that didnot reach the endpoint volume were assigned a TTE value equal to thelast day of the study. Any animal classified as having died from NTR(non-treatment-related) causes due to accident (NTRa) or due to unknownetiology (NTRu) were excluded from TTE calculations and all furtheranalyses. Animals classified as TR (treatment-related) deaths or NTRm(non-treatment-related death due to metastasis) were assigned a TTEvalue equal to the day of death.

Treatment outcome was evaluated from TGD, defined as the increase in themedian TTE in a treatment group compared to the control group:

TGD=T−C,

expressed in days, or as a percentage of the median TTE of the controlgroup:

% TGD=[(T−C)/C]×100

where:

T=median TTE for a treatment group, and

C=median TTE for the designated control group.

Criteria for Regression Responses

Treatment efficacy was determined from the incidence and magnitude ofregression responses observed during the study. Treatment may causepartial regression (PR) or complete regression (CR) of the tumor in ananimal. In a PR response, the tumor volume was 50% or less of its Day 1volume for three consecutive measurements during the course of thestudy, and equal to or greater than 13.5 mm³ for one or more of thesethree measurements. In a CR response, the tumor volume was less than13.5 mm³ for three consecutive measurements during the course of thestudy. An animal with a CR response at the termination of the study wasadditionally classified as a tumor-free survivor (TFS). Animals weremonitored for regression responses.

Toxicity

Animals were weighed daily on Days 1-5, then twice per week untilcompletion of the study. The mice were observed frequently for overtsigns of any adverse, treatment related side effects, and clinical signswere recorded when observed. Individual body weight (BW) loss wasmonitored, and any animal whose weight exceeded the 20% limits foracceptable BW loss was euthanized. Group mean BW loss also was monitoredas per protocol. Dosing was suspended in any group that exceeded the 20%limits for acceptable mean BW loss. If mean BW recovered, then dosingwas resumed in that group, but at a lower dosage or less frequent dosingschedule. Acceptable toxicity for the maximum tolerated dose (MTD) wasdefined as a group mean BW loss of less than 20% during the study andnot more than 10% TR deaths. A death was classified as TR ifattributable to treatment side effects as evidenced by clinical signsand/or necropsy, or was also classified as TR if due to unknown causesduring the dosing period or within 14 days of the last dose. A death wasclassified as NTR if there was no evidence that death is related totreatment side effects.

Statistical and Graphical Analyses

Prism (GraphPad) for Windows 3.03 was used for statistical analyses. Thelogrank test, which evaluates overall survival experience, was used toanalyze significance of the differences between the TTE values of twogroups. Logrank analysis includes data for all animals in a group exceptthose assessed as NTR deaths. Two-tailed statistical analyses wereconducted at significance level P=0.05. The statistical tests were notadjusted for multiple comparisons. Test results are identified as notsignificant “ns” at P>0.05, significant (symbolized by “*”) at0.01<P<0.05, very significant (“**”) at 0.001<P≦0.01, and extremelysignificant (“***”) at P≦0.001. Groups with regimens above the MTD werenot evaluated statistically.

Example 2 Efficacy of TEM8 Antibodies in the HCT116 Colorectal CarcinomaXenograft Model

2.5×10⁶ to 5×10⁶ HCT116 tumor cells were injected subcutaneously intothe flank of mice. Tumor weights were calculated following surgicalremoval. Treatment was initiated when tumor volume reached 60-80 mm³.Treatment groups are shown in Table 3. Dosing volume was 10 mL/kg (0.2mL/20 g mouse).

TABLE 3 Protocol Design for the HCT116-e401 Study Treatment RegimenGroup n Agent Vehicle mg/kg Route Schedule  1^(#) 15 Vehicle HistidineBuffer — IP 3x/wk x 3 2 15 M825 Histidine Buffer 5 IP 3x/wk x 3 3 15M825 Histidine Buffer 15 IP 3x/wk x 3 4 15 M822 Histidine Buffer 5 IP3x/wk x 3 5 15 M822 Histidine Buffer 15 IP 3x/wk x 3 6 15 M863 HistidineBuffer 5 IP 3x/wk x 3 7 15 M863 Histidine Buffer 15 IP 3x/wk x 3 8 15M830 Histidine Buffer 5 IP 3x/wk x 3 9 15 M830 Histidine Buffer 15 IP3x/wk x 3 10  10 Bevaciz- Saline 5 IP 2x/wk x 3 umab ^(#)—Control group,IP—intraperitoneal

FIG. 1A shows the group median tumor volume distribution on day 21 ofthis study. TEM8 Ab (hIgG1) doses were 5 and 15 mg/kg, three times perweek, for 3 weeks. Bevacizumab was dosed at 5 mg/kg, twice per week, for3 weeks. FIG. 1B and FIG. 1D show mean tumor growth and percent bodyweight change for each group, respectively, over the time course of thestudy. FIG. 1C shows the individual times to endpoint for each mouse inthe study. The results show that anti-TEM8 antibodies according to thepresent invention were effective against HCT116 cancer cells in theHCT116 xenograft model.

Example 3 Comparison of TEM8 Antibody Efficacy in UACC Melanoma Model

UACC melanoma cells were cultured and injected subcutaneously into mice.Treatment was initiated with a tumor volume of 50 mm³, and treatmentgroups consisted of one of four TEM8 antibodies (M825, M830, M863, andM822), each dosed at 15 mg/kg, 3 times per week for 3-4 weeks. FIG. 2shows a time course of tumor growth. The results show that TEM 8antibodies, M830 in particular, were effective at slowing tumor growthin the UACC melanoma model.

Example 4 Efficacy of M825 in Xenograft Cancer Models HCT116 ColonCancer Model

Treatment was initiated when tumor volume reached approximately 100 mm³.M825 antibody was given i.v. at a dose of 10 or 30 mg/kg, and theantibody-drug conjugate (ADC) M825-MMAE was given i.v. at 1, 3, 10, or30 mg/kg, 2 times per week for 3 weeks. FIG. 3 shows tumor growth overthe time course of the study. Results showed that the ADC significantlyinhibited tumor growth at 10 and 30 mg/kg in the HCT116 model, eventhough it showed weak cytotoxicity towards HCT116 cancer cells in vitro(data not shown).

OVCAR3 Ovarian Cancer Model

Treatment initiated when tumor volume reached approximately 120 mm³.M825 was given i.v. at 10 mg/kg and M825-MMAE was given i.v. at 1, 3, or10 mg/kg, 2 times per week for 3.5 weeks. The ADC significantlyinhibited tumor growth at 3 mg/kg and regressed tumor growth at 10 mg/kgin this model (FIG. 4). The 10 mg/kg ADC group was redosed on Day 56, 2times per week. No effect was seen with free MMAE at 0.2 mg/kg, a doseequivalent to the maximum amount of MMAE given in the 10 mg/kg ADCgroup.

MDA-MB231 Triple Negative Breast Cancer Model

Treatment was initiated when tumor volume reached approximately 100 mm³.M825 antibody was given i.v. at a dose of 10 mg/kg, and theantibody-drug conjugate (ADC) M825-MMAE was given i.v. at 3 or 10 mg/kg,2 times per week for 3 weeks. The anti-TEM8 ADC treatment led to durabletumor stasis (3 mg/kg) or complete regression (10 mg/kg) in this model,even though it showed only very weak cytotoxicity toward MDA-MB231cancer cells in vitro (data not shown). 50% of animals in the 10 mg/kgADC group were tumor free on Day 35 (FIG. 5).

Example 5 Efficacy of M830 in the H460 Non-Small Cell Lung CarcinomaXenograft Model

Cultured H460 cells were harvested during exponential growth andresuspended in phosphate buffered saline at a concentration of 1×10⁸cells/mL. 1.0×10⁷ tumor cells were injected subcutaneously into theflank of athymic nude mice. Treatment groups are listed in Table 4.Animals were euthanized when tumors reached the pre-determined volumeendpoint of 2000 mm³ or on the final day (Day 88), whichever came first.All regimens were well tolerated, with no treatment related deaths, nonoteworthy clinical signs, and acceptable mean body weight losses. Eachtreatment produced a significant survival difference versus controls(P<0.01). The median TTE for controls was 22.8 days, establishing amaximum possible TGD of 65.2 days (286%) for the 88-day study.

Both combination therapies were statistically superior to thecorresponding monotherapies (P<0.05) but M830 in combination withpaclitaxel was the most active treatment regimen tested in the study.Group 7 produced the maximum TGD and 90% regression responses, two PRsand seven CRs, three of which remained TFS at study end. TheM830/bevacizumab combination produced TGD of 33.2 days (146%) and 40%regression responses which consisted of 1 PR and 3 CRs, all three ofwhich remained TFS at study end.

TABLE 4 Regimen 1 Group n Agent Vehicle mg/kg Route Schedule  1^(#) 10Vehicle PBS — IP 3x/wk x 5 2 10 M830 PBS 5 IP 3x/wk x 5 3 10 M830 PBS 15IP 3x/wk x 5 4 10 Bevaciz- Saline 5 IP biwk x 5/6/biwk x 4 umab 5 10M830 PBS 15 IP 3x/wk x 5/5/ 3x/week x 4 Bevaciz- Saline 5 IP biwk x5/6/biwk x 4 umab 6 10 Paclitaxel 5% EC 30 IV qod x 5/29/qod x 5 7 10M830 PBS 15 IP 3x/wk x 5/5/ 3x/week x 4 Paclitaxel 5% EC 30 IV qod x5/29/qod x 5 ^(#)—Control group, EC—5% ethanol: 5% Cremophor EL in D5W,IP—intraperitoneal, IV—intravenous, qod—every other day

FIG. 6A shows a time course of mean tumor growth throughout the study.FIG. 6B shows a Kaplan-Meier survival plot and FIG. 6C show individualtimes to the endpoint for mice in each group. FIG. 6D shows percent bodyweight change for each group throughout the study. The results show thata combination of M830 and paclitaxel is effective against H460 cancercells in the xenograft model, and that the combination is more effectivethan either agent alone.

Example 6 Efficacy of M830 Alone and in Combination with Gemcitabine,Cisplatin, or Radiation Therapy in H460 Human Non-Small Cell LungCarcinoma Xenograft Model

Treatment:

Treatment began on Day 1 in eight groups of nude mice (n=10) withestablished subcutaneous H460 tumors of 75-126 mm³. The study wasdesigned to evaluate M830 administered i.p. at 15 mg/kg once on Day 1followed by 5 mg/kg three times weekly for five weeks starting on Day 3,alone and in combination with gemcitabine administered i.p. at 120 mg/kgonce every third day for four doses, cisplatin administered i.p. at 6mg/kg once every week for three weeks, or localized radiation at 2 gray(Gray) administered daily for five days followed by a two day break andthen another cycle of five daily doses. The study included thecorresponding gemcitabine, cisplatin and radiation monotherapy groups,as well as a vehicle-treated tumor growth control group. The studyendpoint was a tumor volume of 2000 mm³ or the final day, Day 58,whichever came first. All regimens were well tolerated, with notreatment related deaths, no noteworthy clinical signs, and acceptablemean body weight losses (FIG. 7E). The median TTE for controls was 15.6days, establishing a maximum possible TGD of 42.4 days (272%) for the58-day study.

The M830 and gemcitabine monotherapy treatments resulted in outcomesthat were not statistically different from vehicle-treated controls(P>0.05). FIGS. 7A-B show mean and median tumor volume, respectively,over the time course of the study. Cisplatin and radiation monotherapiesand all three combination therapies produced statistically significantsurvival benefit compared to vehicle-treated controls (see FIGS. 7C-D).In addition, both the M830/gemcitabine and the M830/radiationcombinations were statistically superior to the correspondingmonotherapies (P<0.05). M830 in combination with radiation was the mostactive treatment regimen tested in the study with 238% TGD and four Day58 survivors.

Example 7 Efficacy of M830 Alone and in Combination with Bevacizumab orIrinotecan in the DLD-1 Colorectal Adenocarcinoma Xenograft Model

On the day of tumor implant, each test mouse was injected subcutaneouslyinto the flank with 2.5×10⁶ to 5×10⁶ cultured DLD-1 tumor cells. Tumorweights were calculated following surgical removal. M830 wasadministered i.p. at 5 or 15 mg/kg, three times per week, typically on aMonday, Wednesday, and Friday schedule. Irinotecan was given once perweek at 60 mg/kg i.v. and bevacizumab was dosed at 5 mg/kg i.p., threetimes per week.

FIG. 8 shows a time course of tumor growth throughout the study. Theresults show that the combinations of M830/bevacizumab andM830/irinotecan were effective against DLD-1 cancer cells in thexenograft model, and that the combinations were more effective than anyagent alone.

In a second study, treatment was initiated with a large tumor volume ofapproximately 170 mm³. M830 alone or in combinations with bevacizumab,irinotecan, or both, was given as a loading dose of 20 mg/kg followed by5 mg/kg three times per week. The doses of bevacizumab and irinotecanwere the same as in the first study. An additional group received acombination of bevacizumab and irinotecan without the M830 antibody.

FIG. 9 shows a time course of tumor growth throughout the second study.The results show that all combinations of M830, bevacizumab, andirinotecan were effective against DLD-1 cancer cells in the xenograftmodel, although the triple combination did not outperform theM830/bevacizumab combination therapy.

The study is repeated with one group in each cohort receiving irinotecanat 50 mg/kg once per week i.v. One group in each of the two cohortsreceives a combination of 5 mg/kg M830 three times a week withBevacizumab IP at 5 mg/kg twice per week, fluoropyrimidine at 50 mg/kgonce per week intravenously, and irinotecan at 50 mg/kg once per weekintravenously. Another group in each of the two cohorts receives 15mg/kg M830 three times a week with Bevacizumab IP at 5 mg/kg twice perweek, fluoropyrimidine at 50 mg/kg once per week intravenously, andirinotecan at 50 mg/kg once per week intravenously.

It is expected that the combination of M830, bevacizumab, andfluoropyrimidine-irinotecan will be effective against cancer cells inthe xenograft models and that the combination will be more effective,preferably synergistic compared to any agent alone. The study will berepeated using each of M822, M825, and M863 replacing M830, togetherwith bevacizumab and fluoropyrimidine-irinotecan or other therapeuticagents. It is expected that the combination of M822, M825, or M863 withbevacizumab and fluoropyrimidine-irinotecan or other therapeutic agentswill be effective against cancer cells in the xenograft models and thateach such combination will be more effective, preferably synergistic,compared to any such agent alone.

Example 8 Comparison of M830 Antibody Efficacy in Xenograft Models

Percent tumor reduction and percent tumor reduction vs. combinationpartner for each xenograft model treated with M830 alone or incombination with other chemotherapeutics is shown in Table 5.

TABLE 5 M830 TEM8 Antibody Efficacies in Xenograft Models % TumorReduction % Tumor vs. Reduction vs. combination Cancer CombinationVehicle at Day partner Antibody Model Partner 21* at Day (#) M830 H460Lung Paclitaxel >100% (−61% 94% (32) regression) M830 H460 LungBevacizumab 94.5%   58.5% (32)   M830 H460 Lung N/A 62% N/A M830 DLD1Colon Irinotecan 86% 53% (21) M830 DLD1 Colon Bevacizumab 86% 48% (21)M830 DLD1 Colon N/A 68% N/A M830 UACC N/A 65% N/A Melanoma M830 HCT116N/A 39.5%   N/A Colon *Treatment initiated with tumor volume of 60-80mm³ for H460 and HCT116 studies or about 50 mm³ for DLD1 and UACCstudies; M830 dosed IP, 3 times per week

Example 9 Efficacy of M830 in MC38 Colon Carcinoma Liver MetastasisModel

C57BL/6 mice were challenged with MC38 colon carcinoma liver metastasisby intrasplenic injections of cultured cells. FIG. 10 shows that M830significantly improved the survival of mice in this model (P=0.0001),with a median survival rate of 17 days versus 11 days for vehicletreated mice.

Example 10 Efficacy of TEM8 Antibodies in Combination with MolecularlyTargeted Cancer Pathway Inhibitors

Athymic nude mice are used. On the day of tumor implant, each test mouseis injected subcutaneously in the right flank with 2.5×10⁶ to 5×10⁶DLD-1 cells (cohort 1), or 1×10⁷ cells H460 cells (cohort 2), and tumorgrowth is monitored as the average size approaches the target range of100 to 150 mm³. The endpoint of the study is a tumor volume of 2000 mm³or the last day of the study, whichever comes first.

Monotherapy Treatments

Two groups in each cohort receive M830 at 5 and 15 mg/kg three times aweek. Two groups in each cohort receive the COX2 inhibitor celecoxib at25 and 75 mg/kg daily. (Park et al., 2008). One group in each cohortreceives the non-steroidal anti-inflammatory (NSAID) ibuprofen in chowat 1360 mg/kg. (Yao et al., 2005). One group in each cohort receives theprostaglandin E₂ (PGE₂) synthase inhibitor sulindac sulphide orally at20 mg/kg per day. (Mahmoud et al., 1998).

Combination Treatments

One group in each of the two cohorts receives a combination of 5 mg/kgM830 three times a week with celecoxib orally at 25 mg/kg daily. Anothergroup in each of the two cohorts receives a combination of 5 mg/kg M830three times a week with celecoxib orally at 75 mg/kg daily. A furthergroup in each of the two cohorts receives a combination of 15 mg/kg M830three times a week with celecoxib orally at 25 mg/kg daily. Anadditional group in each of the two cohorts receives a combination of 15mg/kg M830 three times a week with celecoxib orally at 75 mg/kg daily.

One group in each of the two cohorts receives a combination of 5 mg/kgM830 three times a week with ibuprofen in chow at 1360 mg/kg. Anothergroup in each of the two cohorts receives 15 mg/kg M830 three times aweek with ibuprofen in chow at 1360 mg/kg.

One group in each of the two cohorts receives a combination of 5 mg/kgM830 three times a week with sulindac sulphide orally at 20 mg/kg perday. Another group in each of the two cohorts receives 15 mg/kg M830three times a week with sulindac sulphide orally at 20 mg/kg per day.

Results

It is expected that the combination of M830 and celecoxib, M830 andibuprofen, and M830 and sulindac sulphide will be effective againstcancer cells and/or tumor associated stroma and/or tumor vasculature inthe xenograft models and that the combination will be more effective,preferably synergistic, compared to any agent alone. The study will berepeated using each of M822, M825, and M863 replacing M830, togetherwith celecoxib or other COX-2 inhibitors, ibuprofen or other NSAIDs, andsulindac sulphide or other PGE₂ synthase inhibitors. It is expected thatthese combinations will also be effective against cancer cells and/ortumor associated stroma and/or tumor vasculature in the xenograft modelsand that each such combination will be more effective, preferablysynergistic, compared to any such agent alone.

Example 11 Affinity and Pharmacokinetics of TEM8 Antibodies

Table 6 shows Biocore affinity measurements of anti-TEM8 fragmentantigen-binding (Fabs).

TABLE 6 Binding affinities of TEM8 antibodies M830 M825 ka (1/Ms) kd(1/s) K_(D) (M) ka (1/Ms) kd (1/s) K_(D) (M) mTEM8-Fc 7.749E+5 0.0029013.744E−9 1.700E+6 8.942E−5 5.261E−11 hTEM8-AP 6.071E+5 9.533E−4 1.570E−93.050E+6 1.802E−4 5.909E−11

Table 7 shows an in silico assessment of TEM8 antibodies. M830 and M863have more basic V domains, M825 and M822 have more acidic V domains. Theinherent polarity between V and C domains may pose downstreampurification challenges. Sequence analysis identified no potentialglycosylation sites in any CDRs, eliminating one of the biggest risksfor development.

TABLE 7 pI values of TEM8 antibodies pI calculated based on variable pIcalculated based on Antibodies domains only IgG1 M822 5.39 7.91 M8255.47 7.91 M830 10.17 8.75 M863 10.11 8.68

Tables 8 and 9 show pharmacokinetic properties of TEM8 antibodiesaccording to the present invention in mouse for single dose (Table 8)and multiple dose (Table 9) treatments.

TABLE 8 Single dose summary CMAX TMAX AUCI AUCT HALF CLF VZF KEL GroupTreatment (ng/mL) (hr) (hr*ng/mL) (hr*ng/mL) (hr) (mL/hr/kg) (mL/kg)(1/hr) 2 M825 5 mg/kg 46800 3 1310519 1310222 6.03 3.82 33.19 0.1149 3M825 15 mg/kg 144000 3 9239202 8991767 23.77 1.62 55.66 0.0292 4 M822 5mg/kg 42100 3 1611073 1610845 5.75 3.10 25.73 0.1206 5 M822 15 mg/kg182000 24 13523442 13522827 10.50 1.11 16.80 0.0660 6 M863 5 mg/kg 513003 1330847 1330742 8.59 3.76 46.56 0.0807 7 M863 15 mg/kg 355000 310721535 8 M830 5 mg/kg 67500 3 2242485 2242286 8.54 2.23 27.48 0.0811 9M830 15 mg/kg 203000 3 10692742 10692598 14.94 1.40 30.24 0.0464

TABLE 9 Multiple dose summary CMAX TMAX AUCI AUCT HALF CLF VZF KEL GroupTreatment (ng/mL) (hr) (hr*ng/mL) (hr*ng/mL) (hr) (mL/hr/kg) (mL/kg)(1/hr) 2 M825 5 mg/kg 54300 3 1408345 1408201 9.10 3.55 46.61 0.0762 3M825 15 mg/kg 314000 3 21649446 21648879 17.05 0.69 17.05 0.0406 4 M8225 mg/kg 88000 24 4550095 5 M822 15 mg/kg 632000 3 68241171 6657524754.57 0.22 17.31 0.0127 6 M863 5 mg/kg 58600 3 1425735 1425565 14.293.51 72.28 0.0485 7 M863 15 mg/kg 147000 3 7272672 7271700 12.64 2.0637.60 0.0549 8 M830 5 mg/kg 86400 3 4316790 4315918 22.88 1.16 38.240.0303 9 M830 15 mg/kg 330000 3 23053875 23052853 18.70 0.65 17.550.0371

Example 12 M825-MMAE In Vitro and Non-GLP Toxicology Studies

3T3 fibroblasts

3T3 cells were cultured and contractility in the presence of TGFb-1 wasmeasured in control or M825 exposed cells. M825 was shown to block thecontractility of activated 3T3 fibroblasts in vitro (FIG. 11A-B).Contractile forces are thought to contribute to increased cancer cellinvasion.

Toxicology

In a single rising dose phase study design, female (N=3) and male (N=3)rats were given the ADC at intervals of 3 or more days and weremonitored for 2 weeks. Clinical observations, body weight, and necropsyresults were looked at, as well as the clinical pathology andhistopathology for any unscheduled sacrifices. The first dose was 20mg/kg. This dose was considered toxic as 1 out of 6 rats were founddead, 2 with erected fur, and 3 with BW loss. There were dark foci inmany organs, discoloration, platelet decrease, impaired liver and kidneyfunctions, and skin and testis findings. Part of the findings may havebeen due to toxicity of MMAE. The next dose was 40 mg/kg, which wasconsidered lethal as all rats were found dead or moribund by Day 5. 15and 10 mg/kg were chosen for the multiple dose phase study.

In the multiple dose phase study design, 5 male and 5 female rats foreach cohort were given 3 weekly doses at Days 1, 8, and 15, andsacrificed on Day 18. A vehicle control arm was added. In the 15 mg/kggroup, 1 out of 10 animals was found dead (Day 15), 1 moribund (Day 17),2 had decreased activity (Day 15), 2 had erected fur (Day 15), and 3 haddark foci in the lungs. In the 10 mg/kg group, 1 out of 10 was founddead (Day 16), 2 had decreased activity (Day 15), 2 had erected fur (Day15), and 1 had dark loci in the lungs. Both groups also haddiscoloration, skin, thymus, pituitary gland, and lymph node findings,and reduced red blood cell, hemoglobin, and platelet counts.

Example 13 M830 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

Fc mutations which increase ADCC are known in the art. For example,substitutions in the IgG constant region such as S239D, A330L, and I332Eare known to increase binding to the FCy receptor. (see, e.g., Lazar etal., Proc. Natl., Acad. Sci. U.S.A., 103:4005-4010, 2006). In thepresent example, the M830 Fc S239D:I332E double mutant was generated andconfirmed by standard molecular biology techniques (830M: S239D/I332E).FIG. 12A is a schematic showing the antibody binding in ADCC and FIG.12B demonstrates that the Fc mutant form of M830 showed significantlyenhanced binding to FcγRIlla (CD16a).

DOCUMENTS

-   CODACCI-PISANELLI, G., et al. High-dose 5-fluorouracil with    uridine-diphosphoglucose rescue increases thymidylate synthase    inhibition but not 5-fluorouracil incorporation into RNA in murine    tumors. Oncology 62(4): 363-70.-   GEARY, S. M., et al. (2013). The combination of a low-dose    chemotherapeutic agent, 5-fluorouracil, and an adenoviral tumor    vaccine has a synergistic benefit on survival in a tumor model    system. PLOS One 8(6): e67904.-   HE, S., et al. (2013). Discovery of highly potent microsomal    prostaglandin e2 synthase 1 inhibitors using the active conformation    structural model and virtual screen. J Med Chem 56(8): 3296-309.-   ST CROIX, B., et al. (2000). Genes expressed in human tumor    endothelium. Science 289(5482): 1197-1202.

All documents cited in this application are hereby incorporated byreference as if recited in full herein.

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

1. A method for treating or ameliorating the effects of a disease in a subject comprising: administering to a subject in need thereof (a) a therapeutically effective amount of an anti-metabolite agent or analog thereof; and (b) a therapeutically effective amount of a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody comprises: (i) a heavy chain variable region (VH), which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and (ii) a light chain variable region (VL), which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
 2. The method according to claim 1, wherein the subject is a mammal.
 3. The method according to claim 2, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
 4. The method according to claim 2, wherein the mammal is a human.
 5. The method according to claim 1, wherein the disease is characterized by differential expression of tumor endothelial marker 8 (TEM8) membrane antigen.
 6. The method according to claim 1, wherein the disease is characterized by differential expression of tumor endothelial marker 8 (TEM8) membrane antigen on a tumor cell and/or a tumor stromal cell.
 7. The method according to claim 1, wherein the disease is a cancer that differentially expresses TEM8.
 8. The method according to claim 7, wherein the cancer is selected from the group consisting of kidney cancer, colon cancer, lung cancer, liposarcomas, brain cancer, breast cancer, melanoma, liver cancer, head and neck cancer, and prostate cancer.
 9. The method according to claim 1, wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) a VH polypeptide encoded by: (SEQ ID NO: 11) caggtccagctggtgcagtctggggctgaggtgaagaagcctggg acctcagtgaaggtctcctgcaaggttcctggatacaccttcagc agctatgctatcagctgggtgcgacaggcccctggacaagggctt gagtggatgggagggatcatccctatctttggtacaacaaactac gcacagaagttccagggcagagtcacgattaccggggaggaatcc acgagcacagtctacatggagctgagcagcctgagatctgaggac acggccgtgtattactgtgcgagagatacggactacatgtttgac tactggggccagggaaccctggtcaccgtgagctca

and (2) a VL polypeptide encoded by: (SEQ ID NO: 12) tcttctgagctgactcaggaccctgttgtgtctgtggccttggga gagacagtcagtatcacatgccaaggagacaacctcagagacttt tatgcaagctggtaccaacagaagccaggacaggcccctctacta gtcatgtatggtaaaaacaggcggccctcagggatcccagaccga ttctctggctccacctcaggaaacacactttccttgaccatcact ggggctcaggcggaagatgaggctgactattactgtagctcccgg gacaacagtaagcatgtggtgttcggcggggggaccaaggtcacc gtccta.


10. The method according to claim 1, wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) a VH polypeptide encoded by: (SEQ ID NO: 13) caggtccagctggtgcagtctggggctgaggtgaagaagcctggg gcctcagtgaaggtctcctgcaaggtttctggatacaccttcagc agctatgctatcagctgggtgcgacaggcccctggacaagggctt gagtggatgggagggatcatccctatctttggtacagcaaactac gcacagaagttccagggcagagtcacgattaccgcggacgaatcc acgagcacagcctacatggagctgagcagcctgagatctgaggac acggccgtgtattactgtgcgagagatacggactacatgtttgac tactggggccagggaaccctggtcaccgtgagctca

and (2) a VL polypeptide encoded by: (SEQ ID NO: 14) tcttctgagctgactcaggaccctgttgtgtctgtggccttggga gagacagtcagtatcacatgccaaggagacaacctcagagacttt tatgcaagctggtaccaacagaagccaggacaggcccctctacta gtcatgtatggtaaaaacaggcggccctcagggatcccagaccga ttctctggctccacctcaggaaacacactttccttgaccatcact ggggctcaggcggaagatgaggctgactattactgtagctcccgg gacaacagtaagcatgtggtgttcggcggggggaccaaggtcacc gtccta.


11. The method according to claim 1, wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) a VH polypeptide encoded by: (SEQ ID NO: 15) gaggtgcagctggtggagtctgggggaggcgtggtccagcctggg aggtccgtgagactctcctgtgcagcctctggattcaccttcagt acctatactatgcactgggtccgccaggctccaggcaaggggctg gagtgggtggcaattatctcaaatgatggaagcaataagtactac gcagaccccgtgaggggccgattcaccatctccagagacaattcc aagaacacgctgtatctgcaaatgaacagcctgagagctgaggac acggctgtgtattactgtgtacgtggcagcagctggtatcgcgga aattggttcgacccctggggccagggaaccctggtcaccgtgagc tca

and (2) a VL polypeptide encoded by: (SEQ ID NO: 16) gacatccagatgacccagtctccatcctccctgtctgcatctgta ggagacagagtcaccatcgcttgccgggcaagtcagaccattagt aggtatttaaattggtatcagcagaaaccagggaaagcccctaag ctcctgatctatgctgcatccagtttgcaaagtggggtctcatca aggttcagtggcagtggatctgggacagagttcactctcaccatc agcagtctgcagcctgaagattttgcaacttatttctgtcaacag acttacagtcccccgatcaccttcggccaagggacacgactggag attaaacga.


12. The method according to claim 1, wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) a VH polypeptide encoded by: (SEQ ID NO: 17) gaggtgcagctggtggagaccggggctgaggtgaagaagcctggg gcctcagtgaaggtctcctgcaaggcttctggatacaccttcacc ggctactatatgcactgggtgcgacaggcccctggacaagggctt gagtggatgggatggatcaaccctaccagtggtagcacaaactat gcacagaagtttcagggcagggtcaccatgaccagggacacgtcc atcagcacagcctacatggagctgagcgggctgagatctgacgac actgccgtgtattactgtgtgagagatccgggttctcctaagtgg ctggccttcgacccctggggccagggcaccctggtcaccgtgagc tca

(2) a VL polypeptide encoded by: (SEQ ID NO: 18) gacatccagttgacccagtctccatcctccttgtctgcttctgta ggagacagagtcaccatcacttgccgggcaagtcgggccattagt aggtatttaaattggtatcagcagaaaccagggaaagcccctaag ctcctgatctatgctgcatccagtttgcaaagtggggtctcatca aggttcagtggcagtggatctgggacagagttcactctcaccatc agcagtctgcagcctgaagattttgcaacttatttctgtcaacag acttacagtcccccgatcaccttcggccaagggacacgactggag attaaacgt.


13. The method according to claim 1, wherein the anti-metabolite agent or analog thereof is selected from the group consisting of antifolates, purine inhibitors, pyrimidine inhibitors, and combinations thereof.
 14. The method according to claim 1, wherein the anti-metabolite agent or analog thereof is selected from the group consisting of 5-fluorouracil (Tocris Bioscience), tegafur (LGM Pharma), capecitabine (Xeloda) (Roche), cladribine (LGM Pharma), methotrexate (DuraMed Pharmaceuticals, Inc.), pemetrexed (Eli Lilly), hydroxyurea (Bristol-Myers Squibb), 2-mercaptopurine (Sigma-Aldrich), 6-mercaptopurine (Sigma-Aldrich), fludarabine (Ben Venue Laboratories), gemcitabine (Eli Lilly), clofarabine (Genzyme Corp.), cytarabine (Bedford Laboratories), decitabine (Eisai Inc.), floxuridine (Bedford Laboratories), nelarabine (GlaxoSmithKline), pralatrexate (Spectrum Pharmaceuticals), 6-thioguanine (Gate Pharmaceuticals), 5-azacytidine (Pharm ion Pharmaceuticals), doxifluridine (Cayman Chemicals), forodesine (BioCryst Pharmaceuticals), pentostatin (Bedford Laboratories), sapacitabine (Cyclacel Pharmaceuticals, Inc.), thiarabine (Access Pharmaceuticals), troxacitabine (SGX Pharmaceuticals), raltitrexed (AstraZeneca), aminopterin (Sigma Aldrich), carmofur (Santa Cruz Biotechnology, Inc.), azathioprine (GlaxoSmithKline), 6-azauracil (MP Biomedicals, LLC), pharmaceutically acceptable salts thereof, and combinations thereof.
 15. A method for treating or ameliorating the effects of a disease in a subject comprising: administering to a subject in need thereof (a) a therapeutically effective amount of an anti-metabolite agent or analog thereof; and (b) a therapeutically effective amount of a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody comprises: (i) a heavy chain variable region, which comprises an amino acid sequence selected from SEQ ID NO:5; and (ii) a light chain variable region, which comprises an amino acid sequence selected from SEQ ID NO:6.
 16. A method for treating or ameliorating the effects of a disease in a subject comprising: administering to a subject in need thereof (a) a therapeutically effective amount of 5-fluorouracil; and (b) a therapeutically effective amount of a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody comprises: (i) a heavy chain variable region, which comprises an amino acid sequence selected from SEQ ID NO:5; and (ii) a light chain variable region, which comprises an amino acid sequence selected from SEQ ID NO:6.
 17. A composition for treating or ameliorating the effects of a disease in a subject comprising: (a) a therapeutically effective amount of an anti-metabolite agent or analog thereof; and (b) a therapeutically effective amount of a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody comprises: (i) a heavy chain variable region (VH), which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and (ii) a light chain variable region (VL), which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
 18. The composition according to claim 17, wherein the subject is a mammal.
 19. The composition according to claim 18, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
 20. The composition according to claim 18, wherein the mammal is a human.
 21. The composition according to claim 17, wherein the disease is characterized by differential expression of tumor endothelial marker 8 (TEM8) membrane antigen.
 22. The composition according to claim 17, wherein the disease is characterized by differential expression of tumor endothelial marker 8 (TEM8) membrane antigen on a tumor cell and/or a tumor stromal cell.
 23. The composition according to claim 17, wherein the disease is a cancer that differentially expresses TEM8.
 24. The composition according to claim 23, wherein the cancer is selected from the group consisting of kidney cancer, colon cancer, lung cancer, liposarcomas, brain cancer, breast cancer, melanoma, liver cancer, head and neck cancer, and prostate cancer.
 25. The composition according to claim 17, wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) a VH polypeptide encoded by: (SEQ ID NO: 11) caggtccagctggtgcagtctggggctgaggtgaagaagcctggg acctcagtgaaggtctcctgcaaggttcctggatacaccttcagc agctatgctatcagctgggtgcgacaggcccctggacaagggctt gagtggatgggagggatcatccctatctttggtacaacaaactac gcacagaagttccagggcagagtcacgattaccggggaggaatcc acgagcacagtctacatggagctgagcagcctgagatctgaggac acggccgtgtattactgtgcgagagatacggactacatgtttgac tactggggccagggaaccctggtcaccgtgagctca

and (2) a VL polypeptide encoded by: (SEQ ID NO: 12) tcttctgagctgactcaggaccctgttgtgtctgtggccttggga gagacagtcagtatcacatgccaaggagacaacctcagagacttt tatgcaagctggtaccaacagaagccaggacaggcccctctacta gtcatgtatggtaaaaacaggcggccctcagggatcccagaccga ttctctggctccacctcaggaaacacactttccttgaccatcact ggggctcaggcggaagatgaggctgactattactgtagctcccgg gacaacagtaagcatgtggtgttcggcggggggaccaaggtcacc gtccta.


26. The composition according to claim 17, wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) a VH polypeptide encoded by: (SEQ ID NO: 13) caggtccagctggtgcagtctggggctgaggtgaagaagcctggg gcctcagtgaaggtctcctgcaaggtttctggatacaccttcagc agctatgctatcagctgggtgcgacaggcccctggacaagggctt gagtggatgggagggatcatccctatctttggtacagcaaactac gcacagaagttccagggcagagtcacgattaccgcggacgaatcc acgagcacagcctacatggagctgagcagcctgagatctgaggac acggccgtgtattactgtgcgagagatacggactacatgtttgac tactggggccagggaaccctggtcaccgtgagctca

and (2) a VL polypeptide encoded by: (SEQ ID NO: 14) tcttctgagctgactcaggaccctgttgtgtctgtggccttggga gagacagtcagtatcacatgccaaggagacaacctcagagacttt tatgcaagctggtaccaacagaagccaggacaggcccctctacta gtcatgtatggtaaaaacaggcggccctcagggatcccagaccga ttctctggctccacctcaggaaacacactttccttgaccatcact ggggctcaggcggaagatgaggctgactattactgtagctcccgg gacaacagtaagcatgtggtgttcggcggggggaccaaggtcacc gtccta.


27. The composition according to claim 17, wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) a VH polypeptide encoded by: (SEQ ID NO: 15) gaggtgcagctggtggagtctgggggaggcgtggtccagcctggg aggtccgtgagactctcctgtgcagcctctggattcaccttcagt acctatactatgcactgggtccgccaggctccaggcaaggggctg gagtgggtggcaattatctcaaatgatggaagcaataagtactac gcagaccccgtgaggggccgattcaccatctccagagacaattcc aagaacacgctgtatctgcaaatgaacagcctgagagctgaggac acggctgtgtattactgtgtacgtggcagcagctggtatcgcgga aattggttcgacccctggggccagggaaccctggtcaccgtgagc tca

and (2) a VL polypeptide encoded by: (SEQ ID NO: 16) gacatccagatgacccagtctccatcctccctgtctgcatctgta ggagacagagtcaccatcgcttgccgggcaagtcagaccattagt aggtatttaaattggtatcagcagaaaccagggaaagcccctaag ctcctgatctatgctgcatccagtttgcaaagtggggtctcatca aggttcagtggcagtggatctgggacagagttcactctcaccatc agcagtctgcagcctgaagattttgcaacttatttctgtcaacag acttacagtcccccgatcaccttcggccaagggacacgactggag attaaacga.


28. The composition according to claim 17, wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) a VH polypeptide encoded by: (SEQ ID NO: 17) gaggtgcagctggtggagaccggggctgaggtgaagaagcctggg gcctcagtgaaggtctcctgcaaggcttctggatacaccttcacc ggctactatatgcactgggtgcgacaggcccctggacaagggctt gagtggatgggatggatcaaccctaccagtggtagcacaaactat gcacagaagtttcagggcagggtcaccatgaccagggacacgtcc atcagcacagcctacatggagctgagcgggctgagatctgacgac actgccgtgtattactgtgtgagagatccgggttctcctaagtgg ctggccttcgacccctggggccagggcaccctggtcaccgtgagc tca

(2) a VL polypeptide encoded by: (SEQ ID NO: 18) gacatccagttgacccagtctccatcctccttgtctgcttctgta ggagacagagtcaccatcacttgccgggcaagtcgggccattagt aggtatttaaattggtatcagcagaaaccagggaaagcccctaag ctcctgatctatgctgcatccagtttgcaaagtggggtctcatca aggttcagtggcagtggatctgggacagagttcactctcaccatc agcagtctgcagcctgaagattttgcaacttatttctgtcaacag acttacagtcccccgatcaccttcggccaagggacacgactggag attaaacgt.


29. The composition according to claim 17, wherein the anti-metabolite agent or analog thereof is selected from the group consisting of antifolates, purine inhibitors, pyrimidine inhibitors, and combinations thereof.
 30. The composition according to claim 17, wherein the anti-metabolite agent or analog thereof is selected from the group consisting of 5-fluorouracil (Tocris Bioscience), tegafur (LGM Pharma), capecitabine (Xeloda) (Roche), cladribine (LGM Pharma), methotrexate (DuraMed Pharmaceuticals, Inc.), pemetrexed (Eli Lilly), hydroxyurea (Bristol-Myers Squibb), 2-mercaptopurine (Sigma-Aldrich), 6-mercaptopurine (Sigma-Aldrich), fludarabine (Ben Venue Laboratories), gemcitabine (Eli Lilly), clofarabine (Genzyme Corp.), cytarabine (Bedford Laboratories), decitabine (Eisai Inc.), floxuridine (Bedford Laboratories), nelarabine (GlaxoSmithKline), pralatrexate (Spectrum Pharmaceuticals), 6-thioguanine (Gate Pharmaceuticals), 5-azacytidine (Pharm ion Pharmaceuticals), doxifluridine (Cayman Chemicals), forodesine (BioCryst Pharmaceuticals), pentostatin (Bedford Laboratories), sapacitabine (Cyclacel Pharmaceuticals, Inc.), thiarabine (Access Pharmaceuticals), troxacitabine (SGX Pharmaceuticals), raltitrexed (AstraZeneca), aminopterin (Sigma Aldrich), carmofur (Santa Cruz Biotechnology, Inc.), azathioprine (GlaxoSmithKline), 6-azauracil (MP Biomedicals, LLC), pharmaceutically acceptable salts thereof, and combinations thereof.
 31. A composition for treating or ameliorating the effects of a disease in a subject comprising: (a) a therapeutically effective amount of an anti-metabolite agent or analog thereof; and (b) a therapeutically effective amount of a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody comprises: (i) a heavy chain variable region, which comprises an amino acid sequence selected from SEQ ID NO:5; and (ii) a light chain variable region, which comprises an amino acid sequence selected from SEQ ID NO:6.
 32. A composition for treating or ameliorating the effects of a disease in a subject comprising: (a) a therapeutically effective amount of 5-fluorouracil; and (b) a therapeutically effective amount of a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody comprises: (i) a heavy chain variable region, which comprises an amino acid sequence selected from SEQ ID NO:5; and (ii) a light chain variable region, which comprises an amino acid sequence selected from SEQ ID NO:6.
 33. A pharmaceutical composition comprising the composition of claim 17 and a pharmaceutically acceptable diluent or carrier.
 34. A kit comprising the composition of claim 17 packaged together with instructions for its use.
 35. A kit comprising the pharmaceutical composition of claim 33 packaged together with instructions for its use. 